Isolation of male and female gametes, zygotes and proembryos of leek (Allium tuberosum Roxb).

The isolation of male and female gametes is an effective method to study the fertilization mechanisms of higher plants. An osmotic shock method was used to rupture pollen grains of Allium tuberosum Roxb and release the pollen contents, including generative cells, which were mass collected. The pollinated styles were cut following 3 h of in vivo growth, and cultured in medium for 6-8 h, during which time pollen tubes grew out of the cut end of the style. After pollen tubes were transferred into a solution containing 6% mannitol, tubes burst and released pairs of sperm cells. Ovules of A. tuberosum were incubated in an enzyme solution for 30 min, and then dissected to remove the integuments. Following transfer to a dissecting solution free of enzymes, each nucellus was cut in the middle, and squeezed gently on the micropylar end, resulting in the liberation of the egg, zygote and proembryo from ovules at selected stages. These cells can be used to explore fertilization and embryonic development using molecular biological methods for each cell type and development stage.

Omeprazole inhibits α-glucosidase activity and the formation of nonenzymatic glycation products: Activity and mechanism.

In this study, the inhibitory effect and mechanism of omeprazole on α-glucosidase and nonenzymatic glycation were investigated in vitro by using multi-spectroscopic methods and molecular docking. Enzyme kinetic results showed that omeprazole inhibited α-glucosidase in a reversible and noncompetitive manner (IC50= 0.595 ± 0.003 mM). The results from fluorescence quenching and thermomechanical analyses signified that omeprazole reduced the fluorescence intensity of α-glucosidase by forming an omeprazole-α-glucosidase complex primarily driven by hydrogen bonds. Molecular docking further confirmed that hydrogen bonds and hydrophobic forces were the major driving forces for omeprazole binding to α-glucosidase. The nonenzymatic glycation assays revealed that omeprazole had a moderate inhibition against the formation of fructosamine, dicarbonyl compounds, and advanced glycation end products (AGEs). This study provides a new inhibitor of both α-glucosidase and nonenzymatic glycation and provides a practicable candidate for treating diabetes and its complications.

5-Methoxy-2-mercaptobenzimidazole as an efficient inhibitor on tyrosinase: Inhibitory activity and mechanism.

In this study, 5-methoxy-2-mercaptobenzimidazole (5-M-2-MB) was confirmed as an efficient tyrosinase inhibitor by methods of enzyme kinetic, fluorescence quenching, ANS-binding, thermodynamics, energy transfer, and molecular docking in combination. The results proved that 5-M-2-MB significantly inhibited the tyrosinase (IC50 = 60 ± 2 nM) in a reversible and competitive way with the Ki value of 80 ± 1 nM. It quenched the intrinsic fluorescence of tyrosinase through a static mechanism, and caused conformational change of the enzyme by increasing the hydrophobic region. Moreover, this compound could bind to tyrosinase and form 5-M-2-MB-tyrosinase complex by hydrogen bond and hydrophobic interaction. The interactions were generated between 5-M-2-MB and specific amino acid residues (Trp-358, Thr-308, Glu-356, and Asp-357) located on the A chain of tyrosinase. Therefore, this study would offer a theoretical foundation for developing the new tyrosinase inhibitor.

Inhibitory kinetics and mechanism of rifampicin on α-glucosidase: Insights from spectroscopic and molecular docking analyses.

α-Glucosidase is a critical enzyme associated with diabetes mellitus, and the inhibitors of the enzyme play important roles in the treatment of the disease. In this study, the inhibitory effect and mechanism of rifampicin on α-glucosidase were investigated by multispectroscopic methods along with molecular docking technique. The results showed that rifampicin inhibited α-glucosidase activity prominently (IC50 = 135 ±â€¯1.2 µM) in a reversible and competitive-type manner. The fluorescence intensity of α-glucosidase was quenched by rifampicin through forming rifampicin-α-glucosidase complex in a static procedure. And the formation of the rifampicin-α-glucosidase complex was driven spontaneously by hydrophobic forces and hydrogen bonds. The results obtained from molecular docking further indicated that hydrophobic forces were formed between rifampicin and amino acid residues Phe 173, Pro151, and hydrogen bonds were generated by the interactions of rifampicin with residues Ser 180, Asn 414, Gly160, and Gly161 of α-glucosidase. Moreover, it was found that the binding of rifampicin to α-glucosidase could alter the conformation of the enzyme to make it steady, and the binding distance was estimated to be 1.02 nm. Therefore, this study confirmed a novel α-glucosidase inhibitor and possibly contributed to the improvement of newfangled anti-diabetic agent.

Antityrosinase mechanism of omeprazole and its application on the preservation of fresh-cut Fuji apple.

Omeprazole was first evaluated for its antityrosinase activity and preservation of fresh-cut apples. The results obtained from enzymic analyses showed that the omeprazole inhibited tyrosinase activity (IC50 = 40 ±â€¯1.2 µM) with a reversible and competitive mechanism. Fluorescence quenching assays demonstrated that the interaction between omeprazole and tyrosinase was driven by hydrophobic forces and hydrogen bonds in a static procedure. Molecular docking further revealed that hydrogen bonds and hydrophobic forces were generated by omeprazole with the amino acid residues located in the A chain of tyrosinase. Moreover, the results from preservation assays showed that omeprazole could inhibit the activities of polyphenol oxidase (PPO) and peroxidase (POD), prevent the oxidation of total phenolics and flavonoid, thereby delay the browning of fresh-cut apples. Hence, this work identified a novel tyrosinase inhibitor and expands its feasible application as a food preservative.

Condensed Tannins from Longan Bark as Inhibitor of Tyrosinase: Structure, Activity, and Mechanism.

In this study, the content, structure, antityrosinase activity, and mechanism of longan bark condensed tannins were evaluated. The findings obtained from mass spectrometry demonstrated that longan bark condensed tannins were mixtures of procyanidins, propelargonidins, prodelphinidins, and their acyl derivatives (galloyl and p-hydroxybenzoate). The enzyme analysis indicated that these mixtures were efficient, reversible, and mixed (competitive is dominant) inhibitor of tyrosinase. What's more, the mixtures showed good inhibitions on proliferation, intracellular enzyme activity and melanogenesis of mouse melanoma cells (B16). From molecular docking, the results showed the interactions between inhibitors and tyrosinase were driven by hydrogen bond, electrostatic, and hydrophobic interactions. In addition, high levels of total phenolic and extractable condensed tannins suggested that longan bark might be a good source of tyrosinase inhibitor. This study would offer theoretical basis for the development of longan bark condensed tannins as novel food preservatives and medicines of skin diseases.

Antityrosinase and antioxidant properties of mung bean seed proanthocyanidins: Novel insights into the inhibitory mechanism.

This study investigated the structure, antioxidant activity, antityrosinase activity and mechanism of proanthocyanidins from mung bean seed [Vigna radiata (L.) Wilczek]. The structural composition were characterized by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), electrospray ionization-full-mass spectrometry (ESI-Full-MS), and high-pressure liquid chromatography-electrospray ionization-mass spectrometry (HPLC-ESI-MS) techniques. The mung bean seed proanthocyanidins were composed of procyanidins, prodelphinidins, and their rhamnosides. According to enzyme kinetic analysis, these compounds were potent, reversible, and mixed-type inhibitors of tyrosinase. They inhibited the enzyme activity by interacting with enzyme as well as substrates. The results of molecular docking showed that the interaction between mung bean seed proanthocyanidins and tyrosinase was driven by hydrogen bond, hydrophobic and electrostatic interactions. In addition, mung bean seed proanthocyanidins were demonstrated as powerful antioxidants. Therefore, this study confirmed a novel tyrosinase inhibitor and would lay a scientific foundation for their utilization in pharmaceutical and food industries.

Proanthocyanidins purified from fruit pericarp of Clausena lansium (Lour.) Skeels as efficient tyrosinase inhibitors: structure evaluation, inhibitory activity and molecular mechanism.

Fruit pericarp of Clausena lansium (Lour.) Skeels, a food waste, was selected as a raw material for proanthocyanidins. The proanthocyanidins' structures were integrally analyzed using three methods: matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), high performance liquid chromatography electrospray ionization mass spectrometry (HPLC-ESI-MS) and 13C nuclear magnetic resonance (NMR). The results elucidated that these compounds were composed of prodelphinidin (75%) and procyanidin (25%) with a degree of polymerization (DP) up to the 20-mers. They were proved to be remarkable, reversible and mixed competitive inhibitors of tyrosinase according to results from enzyme experiments. The IC50 values were calculated to be 23.6 ± 1.2 and 7.0 ± 0.2 µg mL-1 for the monophenolase and diphenolase activities, respectively. In addition, the proanthocyanidins had a good inhibitory effect on cell proliferation, cellular tyrosinase activity and melanin production of B16 mouse melanoma cells. Chelation between the hydroxyl group on the B ring of the proanthocyanidins and dicopper irons of the enzyme provided one of the feasible mechanisms for the inhibition on the basis of fluorescence quenching and molecular docking analyses. This research would supply the scientific basis to these compounds application in the pharmaceutical, insecticides, and preservative fields.

Inhibition of tyrosinase by cherimoya pericarp proanthocyanidins: Structural characterization, inhibitory activity and mechanism.

In this study, the structure of proanthocyanidins purified from cherimoya (Annona squamosa) pericarp was analyzed by ESI-QTOF-MS and HPLC analyses. The result indicated that these compounds were procyanidin-type proanthocyanidins, consisting mainly of (epi)catechin units linked b y B-type interflavan bonds. The analyses of enzymology showed that the activities of monophenolase and diphenolase of tyrosinase could be powerfully inhibited by the proanthocyanidins. Further researches on the inhibition mechanism demonstrated that they were reversible and competitive inhibitors with the KI value of 27.1±3.1µg/mL. These inhibitors quenched the fluorescence of tyrosinase through a static quenching mechanism and spontaneously formed proanthocyanidins-enzyme complex. Fluorescence changes of proanthocyanidins in the presence of copper ion suggested that the interactions could reduce the fluorescence intensity of these polymers and the molecular docking analysis revealed that copper irons of the enzyme could be chelated by adjacent hydroxyl groups on the B ring of proanthocyanidins. Moreover, proanthocyanidins were proved to be efficient quencher of substrates. These results would lay scientific foundation for their farther application in food and medicine industry.

Rifampicin as a novel tyrosinase inhibitor: Inhibitory activity and mechanism.

In this study, the inhibitory effect and mechanism of rifampicin on the activity of tyrosinase were investigated for developing a novel tyrosinase inhibitor. It was found to have a significant inhibition on the activity of tyrosinase (IC50=90±0.6µM). From the kinetics analysis, it was proved to be a reversible and noncompetitive type inhibitor of the enzyme with the KI value of 94±3.5µM. The results obtained from intrinsic fluorescence quenching indicated that rifampicin could interact with tyrosinase. In particular, the drastic decrease of fluorescence intensity was due to the formation of a rifampicin-enzyme complex in a static procedure which was mainly driven by hydrophobic forces and hydrogen bonding. Moreover, the ANS-binding fluorescence analysis suggested that rifampicin binding to tyrosinase changed the polarity of the hydrophobic regions. Molecular docking analysis further revealed that the hydrogen bonds were generated between rifampicin and amino residues Leu7, Ser52, and Glu107 in the B chain of the enzyme. And the hydrophobic forces produced through the interaction of rifampicin with B chain residues Pro9, Pro14, and Trp106. This work identified a novel tyrosinase inhibitor and potentially contributed to the usage of rifampicin as a potential hyperpigmentation drug.